Difference between revisions of "Team:ZJU-China/Protocol"
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<span class="psg_ttl">Curli</span> | <span class="psg_ttl">Curli</span> | ||
| + | <p><a href="https://static.igem.org/mediawiki/2018/c/c8/T--ZJU-China--protocol1.pdf">Curli Protocol</a> | ||
| + | <p> | ||
| − | + | <!--span class="psg_ttl psg_subtitle">1. Expression</span> | |
| − | <span class="psg_ttl psg_subtitle">1. Expression</span> | + | |
<p>We obtained csgA sequence from the genome of E. Coli MG1655 by PCR and inserted it into Pet26(+). The recombinant plasmid was transfromed into BL21(DE3) and shaken overnight in LB with antibiotics. After reaching an optical density of 0.6~0.8 at 600 nm, the protein expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 25℃. We centrifuged the solution at 12000 rmp for 15mins, and cell pellets were resuspended gently with lysis buffer followed by sonication at the power of 300w for 30mins. The supernatant after centrifugation was kept for future purification. </p> | <p>We obtained csgA sequence from the genome of E. Coli MG1655 by PCR and inserted it into Pet26(+). The recombinant plasmid was transfromed into BL21(DE3) and shaken overnight in LB with antibiotics. After reaching an optical density of 0.6~0.8 at 600 nm, the protein expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 25℃. We centrifuged the solution at 12000 rmp for 15mins, and cell pellets were resuspended gently with lysis buffer followed by sonication at the power of 300w for 30mins. The supernatant after centrifugation was kept for future purification. </p> | ||
<span class="psg_ttl psg_subtitle">2. Purification and Analysis</span> | <span class="psg_ttl psg_subtitle">2. Purification and Analysis</span> | ||
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<p>Stain with coomassie blue for 1h and destain by destaining solution overnight .</p> | <p>Stain with coomassie blue for 1h and destain by destaining solution overnight .</p> | ||
<p style="margin-top: .6em; font-weight: bolder;"></br>• Cango Red Assay</p> | <p style="margin-top: .6em; font-weight: bolder;"></br>• Cango Red Assay</p> | ||
| − | <p>As an amyloid protein, we dyed curli by Cango Red to indicate the expression and characteristic of curli. Centrifuge curli pellet at 12,000g for 5 mins. Resuspend gently by LB and add Cango Red to 30μg/ml CR and incubate at RT for 30 mins. Centrifuge at 14,000rpm for 5mins. Set LB with equal CR as the blank. Measure the absorbance of supernatant at 480nm for CR and 600nm for cell concentration. Compare the expression in CsgA knockout Defect bacteria with that of our recombinant recombinant strain.</p> | + | <p>As an amyloid protein, we dyed curli by Cango Red to indicate the expression and characteristic of curli. Centrifuge curli pellet at 12,000g for 5 mins. Resuspend gently by LB and add Cango Red to 30μg/ml CR and incubate at RT for 30 mins. Centrifuge at 14,000rpm for 5mins. Set LB with equal CR as the blank. Measure the absorbance of supernatant at 480nm for CR and 600nm for cell concentration. Compare the expression in CsgA knockout Defect bacteria with that of our recombinant recombinant strain.</p--> |
Revision as of 16:58, 17 October 2018
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References
[1] Barnhart, Michelle M; Chapman, Matthew R (2006). "Curli Biogenesis and Function". Annual Review of Microbiology. 60: 131-47. doi:10.1146/annurev.micro.60.080805.142106. PMC 2838481. PMID 16704339
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[4] Daniel MC, Astruc D (January 2004). "Gold nanoparticles: assembly, supramolecular chemistry, quantum-size-related properties, and applications toward biology, catalysis, and nanotechnology". Chemical Reviews. 104 (1): 293-346. doi:10.1021/cr030698. PMID 14719978.
[5] Hu M, Chen J, Li ZY, Au L, Hartland GV, Li X, Marquez M, Xia Y (November 2006). "Gold nanostructures: engineering their plasmonic properties for biomedical applications". Chemical Society Reviews. 35 (11): 1084-94. doi:10.1039/b517615h. PMID 17057837.
[6] Heitner-Wirguin, C. (1996). "Recent advances in perfluorinated ionomer membranes: structure, properties and applications". Journal of Membrane Science. 120: 1-33. doi:10.1016/0376-7388(96)00155-X.
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